1. Field of the Invention
The present invention relates to a method for acquiring information, an apparatus for acquiring information and a method for diagnosing disease, and more particularly to a method or apparatus that uses Time of Flight Secondary Ion Mass Spectrometry (TOF-SIMS).
2. Related Background Art
The progress in genomics in recent years has led to a rapid focus on the importance of analysis of proteins that are gene products present in vivo, and on an analysis of a metabolite existing as an end product in vivo, too.
The importance of analyzing the expression and functions of proteins has been indicated before now and analysis methods have developed. A field of study in metabolite is called “metabonomics” or “metabolomics”. Specifically, these methods have been practiced using a combination of:
(1) separation and purification through two-dimensional electrophoresis or high-performance liquid chromatography (HPLC), and
(2) a detection system such as radiometry, optical analysis or mass spectrometry.
Developments in the technology for analyzing proteins include the construction of databases through proteome analysis (exhaustive analysis of protain in a cell), which may be considered the foundation of protein analysis. Devices that are based on databases obtained thereby can be roughly classified into diagnostic devices and devices for development of innovative drugs (screening of drug candidates). However, with respect to each form of application, the conventional methods involve problems with respect to analysis time, throughput, sensitivity, analyzing ability, flexibility and the like. Thus there has been a need for a device that differs to the conventional methods in these respects and which enables miniaturization, enhanced speed and automation. Accordingly, the development of the so-called “protein chip” in which protein is accumulated at a high density is attracting attention as a method that will meet these needs.
A target molecule captured on a protein chip can be detected by the various detection means described below.
In methods involving mass spectrometry (MS) of protein, in recent years Time of Flight Secondary Ion Mass Spectrometry (hereinafter, abbreviated to “TOF-SIMS”) has been used as a high sensitivity mass spectrometry means or surface analysis means. The term “TOF-SIMS” refers to an analysis method for investigating what type of atoms or molecules are present on the outermost surface of a solid sample. TOF-SIMS has the following characteristics. That is, it can detect a trace constituent of 109 atoms/cm2 (amount corresponding to 1/105 of 1 atomic layer of the outermost surface), it can be applied to both organic matter and inorganic matter, it can measure all chemical elements or compounds present on a surface, and it is capable of imaging secondary ions from substances present on a sample surface.
The principles of this method are briefly described below.
In a high vacuum, a high speed pulsed ion beam (primary ion) is applied to the surface of a solid sample, whereby the constituents of the surface are released into the vacuum by a sputtering phenomenon. Ions (secondary ions) having a positive or negative charge that are generated at this time are converged in one direction by an electric field and detected at a position separate from the sample by a fixed distance. When a pulsed primary ion is applied to the solid surface, secondary ions having various masses are generated in accordance with the composition of the sample surface. Since the lighter an ion is the faster it will emit, and conversely, the heavier the ion is the slower it will emit, it is possible to analyze the mass of the generated secondary ions by measuring the time from the generation of secondary ions until the detection (time of flight). Because only secondary ions generated on the outermost surface of a solid sample are released into the vacuum when a primary ion is irradiated on the sample, information on the outermost surface (depth of approximately several nm) can be obtained. Since a primary ion fluence in TOF-SIMS is remarkably small, an organic compound is ionized in a state where it retains its chemical structure, and the structure of the organic compound can be known from the mass spectrum. However, when analyzing artificial high polymers such as polyethylene or polyester, biopolymers such as protein, and the like using TOF-SIMS under normal conditions, small decomposed fragment ionic species are formed and it is thus generally difficult to know the original structure of the sample. When a solid sample is an insulator, the insulator can be analyzed by using an additional pulsed electron beam during gaps in the pulses of irradiation of a primary ion, to thereby neutralize positive charges accumulating on the solid surface. In addition, in TOF-SIMS, it is also possible to generate an ion image (mapping) of the sample surface by scanning a primary ion beam across the sample surface.
Examples of protein analysis using TOF-SIMS include an analysis in which one part of a specific protein is labeled with an isotope such as 15N and imaging of the protein is detected using a secondary ion such as C15N− (A. M. Belu et al., Anal. Chem., 73, 143 (2001)). Further, a study has been reported which estimates the kinds of proteins based on the kinds of fragment ionic species (secondary ions) corresponding to amino acid residues and the relative intensities thereof (D. S. Mantus et al., Anal. Chem., 65, 1431 (1993)). In addition, a study that investigated the limits of detection for TOF-SIMS for protein adsorbed on substrates of various kinds is known (M. S. Wagner et al., J. Biomater. Sci. Polymer Edn., 13, 40 7 (2002)).
Another method of mass spectrometry that employs a protein as a target is a method utilizing field emission (U.S. Pat. No. 5,952,654). In this method, the protein is subjected to covalent bonding or coordinate bonding on a metal electrode through a fissionable open group in accordance with an applied energy, and the protein is introduced into a mass spectrometer by applying a high electric field.
However, since conventional mass spectrometry does not analyze the target substance (e.g. content in cell) itself, but rather takes an eluted protein or the like as its object, there are limitations to the information obtained.
The MALDI method and the SELDI method, an improved version of the MALDI method, are the softest ionization methods of those currently known. They possess excellent characteristics that enable ionization of a protein with a high molecular weight that is susceptible to breakage as it is, and then detection of the parent ion or an ion conforming thereto. This is currently one of the standard ionization methods when analyzing the mass of a protein. However, when applying these methods to mass spectrometry of a protein chip it is difficult to obtain a two-dimensional distribution image (imaging using mass information) of a protein that has a high spatial resolution due to the presence of a matrix substance. More specifically, although the laser beam itself that is the excitation source can be readily condensed to a diameter of about 1 to 2 μm, a matrix substance present in the vicinity of the protein that is the analysis target is vaporized by the laser beam and ionizes, and therefore the spatial resolution obtained when generating a two-dimensional distribution image of the protein by above method is generally only at a level of about 100 μm. Also, in order to scan the condensed laser beam it is necessary to operate a lens and mirror in an intricate manner. In short, when generating a two-dimensional distribution image of a protein by the above method it is difficult to scan the laser beam, and the technique is confined to a method that moves a sample stage on which the sample to be analyzed is placed. When attempting to obtain a two-dimensional distribution image of a protein at a high spatial resolution, a method that moves a sample stage is generally not preferable.
In comparison with the above methods, because the TOF-SIMS method uses a primary ion, convergence and scanning thereof can be easily performed. Thus, a secondary ion image (two-dimensional distribution image) of a high spatial resolution can be obtained, and it is possible to obtain a spatial resolution of a level of about 1 μm. However, with respect to a target substance (e.g. content in cell) on a substrate, when TOF-SIMS measurement is performed under normal conditions, as described above it is generally difficult to know the original structure of the target substance because almost all of the generated secondary ions are small decomposed fragment ionic species. Therefore, for a sample such as a protein chip in which a plurality of proteins are disposed on a substrate, to obtain a secondary ion image (two-dimensional distribution image) of a high spatial resolution with which the kinds of the proteins can be distinguished, it is necessary to employ some kind of contrivance. It is also necessary to employ some kind of contrivance to distinguish metabolic substances in cell by kind. The above method of A. M. Belu et al. is a method in which one part of a specific protein is labeled with an isotope to allow the high spatial resolution of TOF-SIMS to be adequately exploited. However, providing a specific protein with an isotope label for each measurement is not a common technique. In the method of D. S. Mantus et al. that estimates the kinds of proteins from the kinds of fragment ionic species (secondary ions) corresponding to amino acid residues and the relative intensities thereof, difficulties arise when there is a mixture of proteins having similar amino acid structures.
When applying the TOF-SIMS method to tissue from a living organism, for example, a protein molecule, when peptide chains comprising the protein molecule are in a “held state”, the ionization efficiency of secondary ions declines to a large degree. Also, in measurement using TOF-SIMS, since irradiation of a primary ion is conducted in a high vacuum, drying treatment is conducted for the measurement target sample beforehand. If interaction is generated between protein molecules and other biological materials present in the tissue from a living organism at the time of drying treatment and causes aggregation through intermolecular bonding, the ionization efficiency of secondary ions declines still further.
Accordingly, it is preferable to analyze an amount of specific protein molecules present in tissue from a living organism at a high detection sensitivity and with high quantitativeness, and to release the state of peptide chains comprising a protein molecule that are in a “held state” within the tissue to conduct two-dimensional imaging with respect to the distribution state of the abundance of specific protein molecules on a section of the tissue. Further, it is preferable to inhibit interaction between protein molecules and other biological materials, and retain a state whereby secondary ions are generated at a high efficiency from the peptide chains that have been released from the “holding” state. Alternatively, it is preferable to promote and augment generation of secondary ions from a protein molecule present on a section of tissue from a living organism.
Meanwhile, in the TOF-SIMS method, although ion sputtering is performed by irradiating with primary ions the molecule that is the target of analysis, differences in sputtering efficiency arise in accordance with the form of a surface on which primary ion irradiation is conducted. As a result, a difference also arises in the efficiency of generation of secondary ions deriving from the molecule that is the target of analysis. This is a factor that causes variations in the accuracy of analysis. Therefore, it is preferable to also inhibit fluctuations in the efficiency of generation of secondary ions that stem from variations in the forms of surfaces on which primary ion irradiation is conducted. However, the methods disclosed heretofore have not necessarily been adequate in overcoming these points.